Project Summary The aim of this research proposal is to develop a new class of fluorescent nanoparticles for highly sensitive and multicolor imaging of the tumor microenvironment in vivo toward understanding and improving nanoparticle drug delivery. We will focus on semiconductor quantum dots (QDs), which are nanocrystals that exhibit bright fluorescence and unique optical and electronic properties. We have recently designed a new class of quantum dots called 'alloyed quantum wells,' which have equalized fluorescence brightness across a broad spectrum of colors. This novel property is not available from organic dyes, fluorescent proteins, or conventional quantum dots, and will enable quantitative studies of nanoparticle drug delivery to solid tumors. The basic idea is that we can modify the size, surface chemistry, or targeting ligands on these multicolor probes to model nanoparticle drug formulations, which can then be quantitatively compared for uptake and penetration in solid tumors. Because these particles are immensely bright on the single molecule level, intravital microscopy of solid tumors will allow a single-molecule, mechanistic understanding of the rate-limiting steps of drug delivery in a multicolor fashion. This simultaneous multicolor approach is critical for comparisons in the heterogeneous tumor microenvironment, and is not possible with conventional optical probes. In this proposal, we will optically engineer these nanoparticles, develop inert surface coatings for compact sizes and long circulation times in blood, and develop new high-precision bioconjugation strategies based on self- assembly principles. We will use these new probes to image the microscopic processes of targeted-delivery to tumors, concentrating on caveolae-mediated transcytosis, an active transport process that has recently been shown to efficiently pump nanoparticles from the tumor blood vessels into the interstitial tissue. These studies will implement highly relevant orthotopic models of human breast cancer that will ensure clinical significance of the findings. During the mentored phase of this award, the candidate will be co-mentored by Dr. Shuming Nie of Emory University and Dr. Jan Schnitzer of the Proteogenomic Research Institute for Systems Medicine, and will be trained in the use of orthotopic models of human cancer, intravital microscopy techniques, and antibody- based tumor targeting strategies. Both of these mentors are leaders in their respective fields of nanotechnology and cancer biology, which will enable a convergence of expertise to guide this interdisciplinary research project and to facility the transition of the candidate from a mentored postdoctoral fellow to an independent investigator in an academic setting.